Cloning of the prolyl-dipeptidyl-peptidase from Aspergillus oryzae

ABSTRACT

The invention has for object the new recombinant prolyl-dipeptidyl-peptidase enzyme (DPP IV) from  Aspergillus oryzae  comprising the amino-acid sequence from amino acid 1 to amino acid 755 of SEQ ID NO:2 or functional derivatives thereof, and providing a high level of hydrolyzing specificity towards proteins and peptides starting with X-Pro- thus liberating dipeptides of X-Pro type, wherein X is any amino acid. The invention also provides a DNA molecule encoding the enzyme according to the invention, cells expressing the enzyme according to the invention by recombinant technology, an Aspergillus naturally providing a prolyl-dipeptidyl-peptidase activity which has integrated multiple copies of the Aspergillus native promoter which naturally directs the expression of the gene encoding the prolyl-dipeptidyl-peptidase activity, Aspergillus naturally providing a prolyl-dipeptidyl-peptidase activity which is manipulated genetically so that the dppIV gene is inactivated. The invention provides a method for producing the enzyme according to the invention, comprising cultivating the cells of the invention in a suitable growth medium under conditions that the cells express the enzyme, and optionally isolating the enzyme in the form of a concentrate. The invention provides the use of the enzyme or the cells of the invention to hydrolyze protein containing materials. The invention provides the use of an enzyme and/or a cell providing a prolyl-dipeptidyl-peptidase activity, in combination with at least an enzyme providing a prolidase to hydrolyze protein containing materials. In a last further aspect, the invention provides a food product comprising a protein hydrolyzate obtainable by fermentation with at least a microorganism providing a prolyl-dipeptidyl-peptidase activity higher than 50 mU per ml when grown in a minimal medium containing 1% (w/v) of wheat gluten.

TECHNICAL FIELD

The present invention relates to a new recombinantprolyl-dipeptidyl-peptidase from Aspergillus oryzae, a gene encodingthis enzyme, recombinant cells expressing this enzyme, and methods forhydrolysing protein containing materials.

BACKGROUND ART

Hydrolysed proteins, which are widely used in the food industry, may beprepared by hydrolysis of protein material with acid, alkali or enzymes.However, on the one hand, acid or alkaline hydrolysis can destroy theessential amino acids produced during hydrolysis thus reducing thenutritional value, whereas enzymatic hydrolysis rarely goes tocompletion so that the hydrolysed protein contains substantial amountsof peptides.

The filamentous ascomycete Aspergillus oryzae is known to secrete alarge variety of amylases, proteinases and peptidases, the action ofwhich are essential for the efficient solubilisation and hydrolysis ofraw materials (see WO94/25580). Various methods have been usedAspergillus oryzae for the preparation of food products, especiallymethods involving the use of a koji culture.

EP417481 (Nestlé) thus describes a process for the production of afermented soya sauce, in which a koji is prepared by mixing anAspergillus oryzae koji culture with a mixture of cooked soya androasted wheat, the koji is then hydrolysed in aqueous suspension for 3to 8 hours at 45° C. to 60° C. with the enzymes produced duringfermentation of the Aspergillus oryzae koji culture, a moromi is furtherprepared by adding sodium chloride to the hydrolysed koji suspension,the moromi is left to ferment and is then pressed and the liquorobtained is pasteurized and clarified.

EP429760 (Nestlé) describes a process for the production of a flavouringagent in which an aqueous suspension of a protein-rich material isprepared, the proteins are solubilized by hydrolysis of the suspensionwith a protease at pH6.0 to 11.0, the suspension is heat-treated at pH4.6 to 6.5, and the suspension is ripened with enzymes of a koji culturefermented by Aspergillus oryzae.

Likewise, EP96201923.8 (Nestlé) describes a process for the productionof a meat flavour, in which a mixture containing a vegetal proteinaceoussource and a vegetale carbohydrates containing source is prepared, saidmixture having initially at least 45% dry matter, the mixture isinoculated with a koji culture fermented by Aspergillus oryzae and byone or more another species of microorganisms involved in thetraditional fermentation of meat, and the mixture is incubated untilmeat flavours are formed.

Depending on the nature of the protein and the enzymes used forproteolysis, the peptides formed can however have extremely bittertastes and are thus organoleptically undesirable. There is hence a needfor methods of hydrolysing proteins leading to high degree of proteinhydrolysis and to hydrolysates with excellent organoleptic properties.

In addition, in protein rich materials subjected to enzymatichydrolysis, a high level of glutaminase is required to convert glutamineinto glutamic acid which is an important natural taste enhancer (seeWO95/31114). Biochemical analysis of residual peptides in cerealshydrolysed by Aspergillus oryzae, i.e. wheat gluten, shows however thata considerable amount of glutamine remains sequestered in prolinecontaining peptides (Adler-Nissen, In: Enzymatic hydrolysis of foodproteins. Elsevier Applied Sciences Publishers LTD, p120, 1986). Thereis hence a need for methods of hydrolysing proteins leading toliberation of high amount of glutamine.

Among the different proteases known from koji molds, two neutralendopeptidase (Nakadai et al., Agric. Biol. Chem., 37, 2695-2708, 1973),an alkaline endopeptidase (Nakadai et al., Agric. Biol. Chem., 37,2685-2694, 1973), an aspartic protease (Tsujita et al., Biochem. BiophysActa, 445, 194-204, 1976), several aminopeptidases (Ozawa et al., Agric.Biol. Chem., 37, 1285-1293, 1973), several carboxypeptidases (Nakadai etal., Agric. Biol. Chem., 37, 1237-1251, 1970) have been identified andpurified.

More recently a prolyl-dipeptidyl-peptidase activity has been detectedin Aspergillus oryzae, which is an enzyme providing a high level ofhydrolysing specificity towards proteins and peptides starting withX-Pro- thus liberating dipeptides of X-Pro type, wherein X is anyamino-acid (Tachi et al., Phytochemistry, 31, 3707-3709, 1992).

SUMMARY OF THE INVENTION

The present invention has for object the new recombinantprolyl-dipeptidyl-peptidase (DPP IV) from Aspergillus oryzae comprisingthe amino-acid sequence from amino acid 1 to amino acid 755 of SEQ IDNO:2 or functional derivatives thereof.

In a second aspect, the invention also provides a DNA molecule encodingthe enzyme according to the invention.

In a third aspect, the invention provides a cell expressing the enzymeaccording to the invention by recombinant technology.

In a fourth aspect, the invention provides an Aspergillus naturallyproviding a prolyl-dipeptidyl-peptidase activity which has integratedmultiple copies of the Aspergillus native promoter which naturallydirects the expression of the gene encoding theprolyl-dipeptidyl-peptidase activity.

In a fifth aspect, the invention provides an Aspergillus naturallyproviding a prolyl-dipeptidyl-peptidase activity which is manipulatedgenetically so that the dppIV gene is inactivated.

In a sixth aspect, the invention provides a method for producing theenzyme according to the invention, comprising cultivating the cells ofthe invention in a suitable growth medium under conditions that thecells express the enzyme, and optionally isolating the enzyme in theform of a concentrate.

In a seventh aspect, the invention provides the use of the enzyme or thecells of the invention to hydrolyse protein containing materials.

In another aspect, the invention provides the use of an enzyme and/or acell providing a prolyl-dipeptidyl-peptidase activity, in combinationwith at least an enzyme providing a prolidase to hydrolyse proteincontaining materials.

In a last further aspect, the invention provides a food productcomprising a protein hydrolysate obtainable by fermentation with atleast a microorganism providing a prolyl-dipeptidyl-peptidase activityhigher than 50 mU per ml when grown in a minimal medium containing 1%(w/v) of wheat gluten.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Within the following description, the percentages are given by weightexcept where otherwise stated, and the amino acid or nucleotidesequences referred as “SEQ ID NO:” are always presented in the sequencelisting hereafter.

Likewise, the expression “functional derivative of an enzyme” includesall amino acid sequences which differ by substitution, deletion,addition of some amino acids, for instance 1-20 amino acids, but whichkeep their original activities or functions. The selection of afunctional derivative is considered to be obvious to one skilled in theart, since one may easily creates variants of the DPP IV (having theamino acid sequence SEQ ID NO:2) by slightly adapting methods known toone skilled in the art, for instance the methods described by Adams etal. (EP402450; Genencor), by Dunn et al. (Protein Engineering, 2,283-291, 1988), by Greener et al. (Strategies, 7, 32-34, 1994), and/orby Deng et al. (Anal. Biochem, 200, 81, 1992).

In particular, a protein may be generally considered as a derivative toanother protein, if its sequence is at least 80% identical to theprotein, preferably at least 90%, in particular 95%. In the context ofthe present disclosure, the identity is determined by the ratio betweenthe number of amino acids of a derivative sequence which are identicalto those of the DPP IV having the amino acid sequence SEQ ID NO:2(mature sequence 1-755), and the total number of or amino acids of thesaid derivative sequence.

In addition, the term “koji” designates the product of the fermentationwith a koji mold culture of a mixture of a source of proteins and asource of carbohydrates, especially of a mixture of a leguminous plantor of a cooked oleagginous plant and of a cooked or roasted cerealsource, for example of a mixture of soya or cooked beans and of cookedor roasted wheat or rice.

The present invention thus concerns the new prolyl-dipeptidyl-peptidaseenzyme originating from Aspergillus oryzae which comprises theamino-acid sequence from amino acid 1 to 755 of SEQ ID NO:2 orfunctional derivatives thereof. This enzyme may be operably fused to aleader peptide facilitating its secretion in a host where the enzyme isexpressed, for example the Aspergillus oryzae leader peptide having theamino-acid sequence from amino acid −16 to −1 of SEQ ID NO:2 orfunctional derivatives thereof.

A dppIV gene encoding the DPP IV according to the invention may at leastcomprise the coding parts of the nucleotide sequence SEQ ID NO:1, orfunctional derivatives thereof due to the degeneracy of the geneticcode. This sequence is in fact interrupted by a non-coding sequence,called intron, that is spliced during in-vivo transcription (exon I at1836-1841 bp; exon II at 1925-1924 bp; intron at 1842-1924 bp).

A dppIV gene may be obtained in substantially purified form by using themethod described within the following examples from any strain ofAspergillus oryzae. Alternatively, a dppIV gene may be (1) detected alsofrom other genera or species of microorganisms by use of DNA probesderived from the nucleotide sequence SEQ ID NO:1 in a stringenthybridization assay, and (2) recovered by the well known Reverse-PCRmethod by use of suitable primers, for example primers SEQ ID NO:8 and9. In a further aspect, a dppIV gene may also be in-vitro synthesizedand then multiplied by using the polymerase chain reaction, forinstance.

The DNA molecule according to the invention at least comprises a dppIVgene encoding the DPP IV of the invention. This molecule may be in aform of a vector, i.e. a replicative plasmid or an integrative circularor linearized non replicative plasmid. The DNA molecule thus maycomprise, operably linked to the dppIV gene, regulatory sequences nativeto the organism from which derives the gene. Said native regulatorysequences may be the promoter, the terminator, and/or a DNA sequenceencoding a signal sequence that originally regulated the secretion ofthe dppIV gene, such as the Aspergillus orzyzae nucleotide sequencecoding for a signal peptide from nucleotide 1836 to nucleotide 1966 ofSEQ ID NO:1 (without the intron) or functional derivatives thereof dueto the degeneracy of the genetic code. In another embodiment, regulatorysequences may be native sequences that regulate a different gene in thesaid organism of origin or that regulate a different gene in a foreignorganism, for example. A regulatory sequence other than the nativeregulatory sequence will generally be selected for its high efficiencyor desirable characteristic, for example inducibility of a promoter or asequence encoding a peptide signal which will permit secretion of theprotein.

If heterologous expression is preferred, meaning that the genes of theinvention are expressed in another organism than the original host(strain, variety, species, genus, family, order, class or division) theregulatory sequences are preferably derived from an organism similar orequal to the expression host. For example, if the expression host is ayeast cell, then the regulatory sequences will be derived from a yeastcell. The promoter suitable for constitutive expression, preferably in afungal host, may be a promoter from the following genes:glycerolaldhehyde-3-phosphate dehydrogenase, phospho-glycerate kinase,triose phosphate isomerase and acetamidase, for example. Promotersuitable for inducible expression, preferably in a fungal host, may be apromoter from the following genes: endoxylanase IIA, glucoamylase A,cellobiosehydrolase, amylase, invertase, alcohol dehydrogenase andamyloglucosidase. The selection of a desirable regulatory sequenceoperably linked to a sequence of the invention and capable of directingthe expression of the said nucleotide sequence is considered to beobvious to one skilled in the art.

The DNA molecule according to the invention may also comprise aselection marker to discriminate host cells into which the recombinantDNA material has been introduced from cells that do not comprise thesaid recombinant material. Such marker genes are, for example in casefungal expression is preferred, the known ga-2, pyrG, pyr4, pyrA, trpC,amdS or argB genes. The DNA molecule may also comprise at least onesuitable replication origin. Suitable transformation methods andsuitable expression vectors provided with a suitable transcriptionpromoter, suitable transcription termination signals and suitable markergenes for selecting transformed cells are already known in theliterature for many organisms including different bacteria, fungal andplant species. In the event fungal expression is required, theexpression system described in EP278355 (Novartis) may be thusparticularly adapted.

Recombinant koji molds may be obtained by any method enabling a foreignDNA to be introduced into a cell. Such methods include transformation,electroporation, or any other technique known to those skilled in theart.

The invention thus encompasses a recombinant cell comprising the DNAmolecule of the invention, the said cell being able to express the DPPIV of the invention or functional derivatives thereof. These cells maybe derived from the group of fungal, yeast, bacterial and plant cells.Preferably, yeast cells are of the genera Saccharomyces, Kluyveromyces,Hansenula and Pichia, bacterial cells are Gram negative or positivebacteria, i.e. of the genera Escherichia, Bacillus, Lactobacillus,Lactococcus, Streptococcus and Staphylococcus, plant cells are of thevegetable group, and fungal cells are cells that are traditionally usedfor making a koji, such as Aspergillus, Rhizopus and/or Mucor species,notably Aspergillus soyae, Aspergillus oryzae (ATCC 20386), Aspergillusphoenicis (ATCC 14332), Aspergillus niger (ATCC 1004), Aspergillusawamori (ATCC 14331), Rhizopus oryzae (ATCC 4858), Rhizopus oligosporus(ATCC 22959), Rhizopus japonicus (ATCC 8466), Rhizopus formosaensis,Mucor circinelloides (ATCC 15242), Mucor japanicus, Penicillium glaucumand Penicillium fuscum (ATCC 10447). Strains referred by an ATCC numberare accessible at the American Type Culture Collection, Rockville, Md.20852, US. The invention is not limited by such indications which wererather give to enable one skilled in the art to carry out the invention.

Recombinant cells of the invention may comprise the DNA molecule of theinvention stably integrated into the chromosome or on a replicativeplasmid. Among all recombinant cells of the invention thus created, thepresent invention has particularly for object the strains A. oryzae CNCMI-1887, A. oryzae CNCM I-1888 and Pichia pastoris CNCM I-1886.

Preferably, functional copies of the dppIV gene are integrated at apredefined locus of the chromosomal DNA of the host cell.

Accordingly, in order to operably integrate into the chromosome ofprokaryotic cells at least one functional dppIV gene which is not fusedto any promoter, the DNA molecule of the invention may be integrated byusing the process described in EP564966, i.e.,

(1) transforming a host strain organism with a donor plasmid which doesnot replicate in the host strain, wherein the donor plasmid comprises avector backbone and a dppIV gene of the invention operably integrated,without any promoter, into a part of an operon of the host strain,maintaining the frame and the function of the genomic operon of the hoststrain; (2) identifying cointegrate transformants in which the completedonor plasmid is integrated into the genomic operon of the host strain;and (3) selecting an integrant transformant from the cointegratetransformants, wherein the genome of the selected integrant transformantdoes not include the vector backbone of the donor plasmid but doesinclude the dppIV gene, which is operably integrated into the conservedgenomic operon and which is stably maintained and expressed due toselective pressure on the correct functioning of the essential cistronupon growth in a standard medium.

In a second embodiment, in order to stably integrate into the chromosomeof eucaryotic cells only one functional dppIV sequence which is fused toa promoter and a terminator which are native to the host organism, DNAmolecule of the invention may be integrated by slightly adapting theprocess of de Ruiter-Jacobs, Y.M.J.T., Broekhuijsen et al. (A genetransfer system based on the homologous pyrG gene and efficientexpression of bacterial genes in Aspergillus oryzae. Curr. Genet. 16:159-163, 1989), i.e.,

(1) preparing a non-replicative DNA fragment by ligating the dppIV,which is operably linked to a promoter and terminator that are native tothe host organism, downstream a DNA sequence encoding any essentialgene, said essential gene being inactivated by at least a mutationand/or a deletion (this essential gene may be a gene involved in uracilbiosynthesis, such as the pyrG gene in case A. oryzae is used, forexample); (2) selecting a host organism containing the essential genewhich is however inactivated by another mutation(s) or deletion(s); (3)transforming said host organism with the non-replicative DNA fragment;(4) identifying integrate transformants in which the DNA fragment isintegrated so as to restaure the native function of the essential gene;(5) selecting an integrate transformant in which only one DNA fragmentis integrated.

Progeny of an expression host comprising a DNA molecule according to theinvention is also included in the present invention. Accordingly, apreferred embodiment of the invention is directed to a cell comprising arecombinant DNA molecule of the invention in any of the embodimentsdescribed above, wherein the said cell is able to integrate the DPP IVinto the cell wall or the cell membrane or secrete the enzymes into theperiplasmic space or into the culture medium. The secreting route to befollowed by the recombinant protein according to the invention willdepend on the selected host cell and the composition of the recombinantDNA according to the invention. Most preferably, however, the proteinwill be secreted into the culture medium. To this end, the cellaccording to the invention may comprise a recombinant dppIV gene furtheroperably linked to a DNA encoding a foreign leader sequence (pre orprepro), for example.

Cells over-expressing the DPP IV of the invention are preferably chosen,especially Aspergillus cells capable of providing at least 50 mU,especially at least 100 mU, of DPP IV activity per ml of supernatantwhen grown in a minimal medium containing 1% (w/v) of wheat gluten, suchas the MMWG medium.

These cells may be obtained by incorporation of the DNA molecule of thepresent invention in an expression host, said DNA molecule comprisingone or more regulatory sequences which serve to increase expressionlevels of the protein(s) of the invention.

The over-expression can be further achieved by introducing multicopiesof the DNA molecule of the invention, for example. Surprisingly,Aspergillus cells having integrated multiple recombinant functionaldppIV genes of the invention may provide a DPP IV activity per ml ofsupernatant which is more than it should have been compared to thenumber of integrated copies, probably due to the titration of anegatively acting transcription factor. As an example, the Aspergillusoryzae transformant 6 of the following example 1 was deposited under theBudapest Treaty at the CNCM where it receives the deposit number CNCMI-1888.

In addition, it has also been shown that over-expression of the DPP IVmay be achieved in Aspergillus species naturally providing aprolyl-dipeptidyl-peptidase activity, by integrating multiple copies ofthe Aspergillus native promoter which naturally directs the expressionof the gene encoding the prolyl-dipeptidyl-peptidase activity. Thepromoter region of Aspergillus oryzae contained in the nucleotidesequence from nucleotide 1 to nucleotide 1835 of SEQ ID NO:1 is ofparticular interest for this purpose. As an example, the Aspergillusoryzae transformant B2 of the following example 4 was deposited underthe Budapest Treaty at the CNCM where it receives the deposit numberCNCM I-1887.

The invention is also directed to a process for producing the DPP IV ofthe invention comprising, providing recombinant cells according to theinvention in a suitable growth medium under conditions that the cellsexpress the DPP IV, and optionally isolating the said recombinantprotein(s) in the form of a concentrate. The selection of theappropriate medium may be based on the choice of expression host and/orbased on the regulatory requirements of the DNA recombinant material.Such media are well-known to those skilled in the art.

After fermentation, the cells can be removed from the fermentation brothby centrifugation or filtration. Depending on whether the host cellshave secreted the DPP IV of the invention into the medium or whether theDPP IV are still connected to the host cells in some way either in thecytoplasm, in the periplasmic space or attached to or in the membrane orcell wall, the cells can undergo further treatment to obtain therecombinant protein. In the latter case, where the recombinant enzyme isstill connected to the cells, recovery may be accomplished by rupturingthe cells for example by high pressure, sonication, enzymatic digestionor simply by cell autolysis followed by subsequent isolation of thedesired product. The DPP IV can be separated from the cell mass byvarious methods, such as ultrafiltration, and then subsequentlyprecipitated with an organic solvent. The isolated DPP IV may be furtherpurified by conventional methods such as precipitation and/orchromatography.

The present invention also relates to the use of the purified DPP IV orthe above mentioned cells to hydrolyse protein containing materials,such as mixtures of a source of proteins and a source of carbohydrates,especially of a mixture of a leguminous plant or of a cooked oleaginousplant and of a cooked or roasted cereal source, for example of a mixtureof soya or cooked beans and of cooked or roasted wheat or rice.Compositions containing wheat gluten are particularly adapted for thepurpose of the present invention, since considerable amount of glutamineremains sequestered in proline containing peptides when wheat gluten ishydrolysed by traditional koji cultures.

To obtain a satisfactory degree of hydrolysis, the purified DPP IV maysuitably be added to the proteinaceous material in a amount of 0.05-15Unit/ 100 g of protein, in particular 0.1-8 Unit/100 g of protein. Theincubation may be performed at a pH from between about 4 and about 10,preferably between about 5 and about 9. The incubation may be performedat any convenient temperature at which the enzyme preparation does notbecome inactivated, i.e. in the range of from about 20° C. to about 70°C.

In addition, in the event one may try, after or during hydrolysis withDPP IV, to further liberate as much as possible glutamine linked toproline residues, the present invention provides a method in which theDPP IV of the invention is used in combination with at least an enzymeproviding a prolidase activity that is to say an enzyme which has a highlevel of specificity towards dipeptides of the X-Pro type (Ezespla etal., Ap. Env. Microb., 63, 314-316, 1997; Such kind of enzyme is alreadyavailable from Sigma: E.C. 3.4.13.9).

In a further aspect, the present invention relates to a food productcomprising a protein hydrolysate obtainable by fermentation with atleast a microorganism providing a prolyl-dipeptidyl-peptidase activityhigher than 50 mU per ml when grown in a minimal medium containing 1%(w/v) of wheat gluten.

Important food products of the present invention is an ingredient of amother milk substitute for infants, or a hydrolysed vegetable proteiningredient, i.e. a koji. Indeed, if the DPP IV activity (enzyme ormicroorganism) is combined with other proteolytic activities (enzymes ormicroorganisms), i.e. typically if Pichia pastoris CNCM I-1886 orAspergillus oryzae CNCM I-1887 or CNCM I-1888 or enzyme purificatesthereof are used, high degree of hydrolysis may be obtained leading to anon-bitter flavour and a significantly lower allergenicity thanunhydrolysed proteins. The milk substitute may be further formulated insubstantially the same way as that indicated in the prior literature forproducts of this type (cf. EP 96202475.8).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention, in addition to those described herein, will become apparentto those skilled in the art from the foregoing description andaccompanying figures. Such modifications are intended to fall within thescope of the claims. Various publications are cited herein, thedisclosures of which are incorporated by reference in their entiretiesto the extent necessary for understanding the present invention. DNAmanipulation, cloning and transformation of bacteria cells are, exceptwhere otherwise stated, carried out according to the textbook ofSambrook et al. (Sambrook et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, U.S.A., 1989). Theseexamples are preceded by a brief description of the plasmids and strainsused, and by the composition of various media. The strains A. oryzaeTK3, A. oryzae transformant 6 (example 1), A. oryzae transformant B2(example 4), Pichia pastoris containing pKJ 115 (example 3) weredeposited under the Budapest Treaty, at the Collection Nationale deCulture de Microorganismes (CNCM), 25 rue du docteur Roux, 75724 Paris,France, on Jun. 24, 1997, where they receive respectively the depositnumbers CNCM I-1882, CNCM I-1888, CNCM I-1887 and CNCM I-1886. Allrestrictions as to the availability of these deposits will be withdrawnupon first publication of this application or another application whichclaims benefit of priority to this application.

Strains and Plasmids

Aspergillus oryzae 44 and TK3 originate from the Nestlé straincollection. However other wild type Aspergillus oryzae strains may alsohave been used in the context of the following examples.

A. oryzae NF1 derived from TK3 by targeted disruption (uridineauxotrophe).

Aspergillus nidulans 033 (biA 1, argA1) can be obtained through FungalGenetic Stock Center, Glasgow, and is used as a source of pyrG (GenBankaccession number M19132) gene. However other wild type Aspergillusnidulans strains may also have been used in the context of the followingexamples.

The Pichia pastoris (Invitrogen Inc., US)

Plasmid pMTL21-H4.6 containing the Aspergillus fumigatus dppIV gene canbe provided by the Institut Pasteur, Paris, France (Beauvais et al., Anhomolog of the CD26 is secreted by the human pathogenic fungusAspergillus fumigatus, Infect. immun. In press., 1997; GenBank EMBL,accession number: V87950).

Plasmid pNFF28 contains the A. oryzae TK3 pyrG gene (GenBank EBI/UK,accession number: Y13811).

Plasmids pMTL20 (Chambers et al., Gene, 68, 139-149, 1988; GenBank EMBL,accession number: M21875), pNEB 193 (Biolabs, New England) andpBluescriptSK⁻ (Stratagene, US) were used in subcloning procedures.

Plasmid pCL1920b is a derivative of plasmid pCL1920 (Lerner and Inouye,Nucleic Acids Research, 18, 4631, 1990) in which the multiple cloningsite was modified to include a SmaI site and a EcoRI site between theBamHI and SalI sites.

The P. pastoris expression vector pKJ115 was constructed by cloning theexpression cassette of pPIC9 (Invitrogen) in pCL1920b. In pKJ115 theexpression cassette of pPIC9 is flanked by two SmaI sites forlinearisation of the DNA, before transformation of P. pastoris.

Growth Media

Aspergillus oryzae can grow on the minimal medium (MM) preparedaccording to Pontecorvo et al. (Adv. Genet., 5, 141-239, 1953).

Aspergillus oryzae NF1 is grown at 35° C. on MM containing 10 mM NaNO₃as a nitrogen source and 10 mM uridine.

MMWG contains MM plus 1% (w/v) of wheat gluten (WG) (Sigma),

MMWGH contains MM and 0.1% (w/v) WG (Sigma) plus 0.1% (w/v) WGhydrolysate prepared hydrolysing non-vital wheat gluten powder(Roquette, France) with Alcalase 2.4 L (Novo Nordisk, Denmark).Hydrolysis is conducted at 20% (w/w) substrate concentration and anenzyme to substrate ratio (E/S) of 1:50 (by weight of protein) for 6 hat 60° C. and constant pH of 7.5 (pH stat). Alcalase is then heatinactivated at 90° C. for 10 min. After centrifugation of thehydrolysate, the supernatant is lyophilised to give WGH and stored atroom temperature. WGH contains mainly peptides and only minimal amountsof free amino acids. Peptide mass distribution in WGH is from 200 to10′000 Da, determined by size-exclusion chromatography on a SuperdexPeptide column.

P. pastoris can grow on RDB (Regeneration Dextrose Base): 1M sorbitol,1% (w/v) dextrose, 1.34% (w/v) yeast nitrogen base (YNB), 4×10⁻⁵% (w/v)biotine, 5×10⁻³% aa (i.e. 5×10⁻³% (w/v) of each L-glutamic acid,L-methionine, L-lysine, L-leucine and L-isoleucine.

MMM (Minimal Methanol Medium): 1.34% (w/v) YNB, 4×10⁻⁵% (w/v) biotine,0.5% (w/v) methanol.

BMGY (Buffered minimal Glycerol-complex Medium): 1% (w/v) yeast extract,2% (w/v) peptone, 10 mM potassium phosphate pH 6.0, 1.34% (w/v) YNB,4×10⁻⁵% (w/v) biotine, 1% (w/v) glycerol.

BMMY: (Buffered minimal Methanol-complex Medium): 1% (w/v) yeastextract, 2% (w/v) peptone, 10 mM potassium phosphate pH 6.0, 1.34% YNB,4×10⁻⁵% (w/v) biotine, 0.5 (w/v) % methanol.

EXAMPLES Example 1

Cloning of the dppIV

Screening of a genomic library: a genomic DNA library was prepared usingthe DNA from A. oryzae 44 and screened with a DNA fragment containingthe dppIV gene of Aspergillus fumigatus (Beauvais et al., GenBank EMBL,accession number: V87950).

For this purpose, the isolation of the genomic DNA was performedaccording to a modified protocol of the method described by Raeder andBroda (Let. appl. Microbiol., 1, 17-20, 1985). Mycelium was harvested byfiltration, immediately frozen in liquid nitrogen and lyophilised. Itwas then grinded to a fine powder using a mortar and pestle. 200 mg ofthe powdered mycelium was resuspended in 2.5 ml of extraction buffer(200 mM Tris-HCl pH 8.5 150 mM NaCl, 25 mM EDTA, 0.5% SDS) and thesolution was extracted with 1.75 ml extraction buffer-equilibratedphenol and 0.75 ml of chloroform/isoamylalcohol (24:1, v/v). The mixturewas centrifuged (20 min, 3000 g). The aqueous phase was retrieved andincubated with 125 μl of RNAse A (Boehringer) solution (10 mg/ml) for 10min at 37° C. 1.25 ml of 2-propanol (Merck) were then added. The pelletwas washed with 70% ethanol and finally resuspended in 500 ml of TEbuffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). 500 μl of 2×QBT (1.5 M NaCl,100 mM MOPS, 30% ethanol, pH 7.0) were added to the sample which wasthen applied to a “Genomic-tip” (Qiagen), rinsed and eluted asrecommended by the supplier.

The genomic DNA was then partially digested with Sau3A, and DNAfragments of 12-20 kb were isolated from low melting agarose (Biorad).These fragments were inserted into bacteriophages using the λ EMBL3BamHI arm cloning system (Promega, US).

40000 recombinant plaques of the A. oryzae 44 genomic library in λ EMBL3were immobilised on nylon membranes (Genescreen, Dupont). These filterswere probed, with the ³²P-labelled 2.3 kb dppIV insert of pMTL21-H4.6amplified by PCR in a 5×SSC solution containing 20% formamide, 1% sodiumdodecyl sulfate (SDS), and 10% dextran sulfate at 42° C. for 20 h.Labelling of DNA was performed using a random-primed DNA labelling kit(Boehringer) and (α³²P)-dATP. The membranes were exposed to X-ray filmafter two 20 min washes in 3×SSC-1% SDS at 40° C.

Ten positive clones were isolated and purified. Restriction enzymeanalysis of purified bacteriophage DNA revealed that the clones carriedsimilar but not identical DNA fragments. By Southern analysis, the dppIVgene was assigned to an ApaI-EcoRV 4.8 kb fragment which was subclonedinto pBluescriptSK⁻, creating the plasmid pNFF125.

Checking of functionalities: plasmid pNFF125 was introduced into A.oryzae NF1 by cotransformation with plasmid pNFF28, carrying the pyrGgene for selection of transformants.

For this purpose, A. oryzae NF1 was grown overnight in MM with 50 mMglucose, 5 mM glutamine and 10 mM uridine. The mycelium was harvested bysterile over cheese cloth filtration, washed once with sterile doubledistilled water and once with K0.8MC (20 mM MES-HCl pH 5.8, 0.8 M KCl 50mM CaCl₂). 2 g of mycelium were resuspended in 20 ml of a filtersterilised 5 mg/ml solution of Novozyme 234 in K0.8MC. The myceliumsuspension was incubated at 30° C. for 2 hours with gentle agitation(120 rpm). The protoplasts were liberated from the mycelium by gentleresuspension with a pipette, washed twice with 20 ml of S1.0TC (10 mMTris-HCl pH 7.5, 1 M Sorbitol, 50 mM CaCl₂) and were resuspended at afinal concentration of 10⁸/ml in S1.0TC. 20 ml of DNA was mixed with 200ml of protoplasts and 50 ml of 25% PEG 6000 (BDH) in 10 mM Tris-HCl pH7.5, 50 mM CaCl₂ and incubated for 20 min on ice. To this mixture, 2 mlof 25% PEG 6000 in 10 mM Tris-HCl pH 7.5, 50 mM CaCl₂ were added, gentlymixed and incubated for 5 min at room temperature. 4 ml of S1.0TC wasadded and 1.0 ml aliquots were mixed with 5 ml of 2% low melting pointagarose (Sigma) SMM (MM plus 50 mM glucose and 5 mM glutamine,osmotically stabilised with 1.0 M sucrose) and plated onto SMM agar(Difco).

Ninety-five pyrG⁺ transformants were screened for DPP IV activity afterincubation (2 days, 30° C.) on MMWGH. For this purpose, spores oftransformants were resuspended in SP2 buffer (20 mM KH₂PO₄ adjusted topH 2.0 with HCl and 0.9% NaCl) in microtiter plates and replica platedonto Petri dishes containing MMWGH covered by a Whatman filter (Chr1).The plates were incubated for 2 days at 30° C. DPP IV activity wasdetected on the filter according to Lojda (Histochemistry, 54, 299-309,1977) and Aratake et al. (Am. J. Clin. Pathol., 96, 306-310, 1991).Filters were reacted with a solution of 3 mg glycyl proline 4-βnaphthylamide (Bachem) in 0.25 ml N,N-dimethylformamide (Merck) and 5 mgo-dianisidine, tetrazotized (Sigma) in 4.6 ml 0.1 M sodium phosphatebuffer pH 7.2 for 10 min at room temperature. Endoproteolytic enzymeactivity was also measured with resorufin-labeled casein according toBoehringer method description supplied with the substrate(Resorufin-labeled casein, Cat.No. 1080733). Leucine aminopeptidase anddipeptidyl peptidase IV activities were determined by UV spectrometrywith synthetic substrates Leu-pNa and Ala-Pro-pNa (Bachem, Switzerland),respectively, according to Sarath et al. (Protease assay methods inProteolytic enzymes: a practical approach, IRL Press, Oxford, 1989). 10mM substrate stock solution in dimethylsulfoxide (DMSO) was diluted with100 mM sodium phosphate buffer, pH 7.0, to a final concentration of 0.5mM. 20-100 μl culture medium supernatant was added and reactionproceeded for up to 60 min at 37° C. A control with blank substrate andblank supernatant was done in parallel. The release of the chromophoricgroup 4-nitroaniline (ε: 10′500 M⁻¹cm⁻¹) was measured at 400 nm andactivities were expressed as mU/ml (nmol/min/ml).

Results show that sixteen transformants exhibited a clearly increasedstaining compared to the wild type. Seven transformants numbered 1 to 7were selected because of their high DPP IV activity. Southern blots ofthem confirmed that the increase in the activity was due to theintegration of multiple copies of the 4.8 kb ApaI-EcoRV fragment in thegenome of the transformants. From densitometric scans of these Southernblots, it was estimated that in transformant 1, at least 4 additionalcopies had been functionally integrated into the genomic DNA, while, intransformant 6, they were at least 9 additional copies.

To quantify the increase of DPP IV activity in the transformants 1 and6, these were grown in parallel with control A. oryzae NF1 pyrG⁺, for 7days at 30° C. without shaking in 100 ml liquid MMWG. Analyses of thesupernatants are shown in Table 1. Transformants 1 and 6 showed a DPP IVactivity of at least 8 and 17 times more, respectively, than A. oryzaeNF1 pyrG⁺ transformant, while their leucine-aminopeptidase (LAP) andendopeptidase (ENDO) activities remain unchanged. These data stronglysuggested that pNFF125 contained a functional dppIV gene. In addition,when a functional gene was introduced, the DPP IV activity increasedmore than it should have been compared to the number of integratedcopies. The difference might also come from the titration of anegatively acting transcription factor (repressor).

TABLE 1 DPP IV [mU/ml] LAP [mU/ml] ENDO [mU/ml] NF1 pyrG⁺ 8.7 1.6 2.9Transformant 1 73.9 1.7 3.3 Transformant 6 160.6 1.9 3.1

Characterisation of the DPP IV: culture broth from prolyl dipeptidylpeptidase overproducing transformant 6 and the control A. oryzae NF1pyrG⁺ were analysed by SDS-PAGE. No single band in the prolyl dipeptidylpeptidase-overproducing strain stained more intensely than the A. oryzaeNF1 pyrG⁺ control. However, a broad smear was visible in the regionaround 95 kDa of the prolyl dipeptidyl peptidase-overproducing strain,but not in the A. oryzae NF1 pyrG⁺ control. This aberrantelectrophoretic behaviour might be caused by glycosylation of theenzyme. Therefore, culture broths were treated with N-glycosidase F andreanalysed. In the deglycosylated samples a band of 85 to 90 kDaappeared in the control NF1 pyrG⁺ and in the prolyl dipeptidyl peptidaseoverproducing transformant. A sample of the N-glycosidase F treatedculture medium of transformant 6, corresponding to 100 mU prolyldipeptidyl peptidase activity, was loaded onto a preparative gel andblotted onto an Immobilon p^(SQ) membrane. The putative prolyldipeptidyl peptidase band was excised and analysed by automated Edmandegradation. The N-terminal sequence of the mature protein wasdetermined to be Leu-Asp-Val-Pro-Arg- . . .

Sequencing of the ApaI-EcoRV fragment: the 4.8 kb fragment from pNFF125was sequenced on both strands. The nucleotide sequence of the dppIV genewas determined, on a Licor model 4000 automatic sequencer. IRD41labelled primer having the nucleotide sequence SEQ ID NO:3 was used forsequencing both strands of partially overlapping subclones by thedideoxynucleotide method of Sanger et al. (Proc. Natl. Acad. Sci. USA,74, 5463-5467, 1977). The DNA sequence analysis was performed by usingthe GCG Computer programs (Devereux et al., Nucl. Acids Res., 12,387-395, 1987).

The position of transcription start sites were mapped by primerextension. Additionally the position of exons and intron were determinedby RT-PCR. For this purpose, total RNA was isolated from the A. oryzaeTK3 mycelia cultured overnight on MMWGH, using the “RNeasy Total RNAPurification kit” (Qiagen). Reverse transcriptase PCR (RT-PCR) wasperformed using the “1st strand cDNA synthesis kit for RT-PCR”(Boehringer). 10 μg of total RNA, 1×reaction buffer (10 mM Tris, 50 mMKCl pH 8.3), 5 mM MgCl₂, 1 mM deoxynucleotide mix, 1.6 μg oligo-p(dT)₁₅primer, 50 units RNAse inhibitor, 10 units AMV Reverse transcriptasewere mixed and incubated 25° C. 10 min, 42° C. 60 min, 75° C. 5 min and4° C. 5 min. 1 μl, 2 μl and 3 μl of the obtained cDNA, 2 mM ofoligonucleotides and 250 mM dNTPs (Boehringer) were dissolved in 50 mlof 1×PCR buffer (20 mM Tris-HCl pH 8.55, 16 mM (NH₄)₂SO₄, 2.5 mM MgCl₂,150 mg/ml BSA). To each reaction 1.5 unit of Taq-polymerase (Biotaq)were added as well as one drop of Nujol mineral oil (Perkin Elmer). Thetargeted region of the dppIV gene was amplified, using a Stratagene RoboCycler gradient 40, with the primer pair SEQ ID NO:4 and SEQ ID NO:5.The reaction mixtures were subjected to 2 cycles of 1 min 98° C., 2 min56° C. and 2 min 72° C., followed by 27 cycles of 1 min 94° C., 1 min56° C. and 2 min 72° C. and 1 1 cycle of 1 min 94° C., 1 min 56° C. and10 min 72° C. The gel purified PCR products were recovered with Qiaex II(Qiagen) and directly ligated into the pGEM-T vector (Promega) accordingto the instructions of the manufacturer, to generate plasmid pNFF137.

Results show that the open reading frame (ORF) is split by a 83 bpintron into 2 exons. Furthermore, the 16 aa long N-terminal secretorysignal sequence was identified by homology with the A. fumigatussequence which corresponds well to the signal sequence rule described byVon Heijne (Nucleic Acids Res., 14, 4683-4690, 1986). The dppIV gene hasthe nucleotide sequence SEQ ID NO:1, and encodes a mature protein of 755aa with a deduced molecular weight of 85.4 kDa (see SEQ ID NO:2). Thesignal sequence of dppIV runs from position 1835 (ATG) to 1966 andincludes the intron. The mature protein starts at position 1967 with theamino acid sequence LeuAspValProArg as confirmed by Edman degradation.The exon 1 starts at position 1836 and ends at position 1841; intronstarts at position 1842 and ends at position 1924; exon II starts atposition 1925 and ends at position 4231.

Example 2

Disruption of the dppIV Gene

In order to determine if the cloned dppIV gene was exclusivelyresponsible for the DPP IV activity observed onto MMWGH, it wasdisrupted.

As heterologous selection marker, to prevent targeting of the disruptingconstruct to the pyrG locus, the A. nidulans pyrG gene was amplifiedfrom A. nidulans 033. To do so, the sequences between position 500 and2342 of the pyrG gene (Oakley, et al., Gene, 61, 385-399, 1987) wereamplified by PCR. 200 ng A. nidulans 033 genomic DNA, 2 mM ofoligonucleotides and 250 mM dNTPs (Boehringer) were dissolved in 50 mlof 1×PCR buffer (20 mM Tris-HCl pH 8.55, 16 mM (NH₄)₂SO₄, 2.5 mM MgCl₂,150 mg/ml BSA). To each reaction 1.5 unit of Taq-polymerase (Biotaq)were added as well as one drop of Nujol mineral oil (Perkin Elmer). Thetargeted region was amplified, using a Stratagene Robo Cycler gradient40, with the primer pairs SEQ ID NO:6 and SEQ ID NO:7. The reactionmixtures were subjected to 30 cycles of 1 min 95° C., 1 min 52° C. and 3min 72° C. The gel purified 1.8 kb PCR product was recovered with QiaexII (Qiagen) and cloned into pGEM-T (Promega), according to theinstructions of the manufacturer, to give pNFF39.

In parallel, a mutant allele of dppIV was generated from pNFF 125 byreplacing the internal 1.5 kb NcoI fragment with the 1.8 kb NcoIfragment from pNFF39, creating pNFF129.

ApaI-EcoRV digested pNFF129 was introduced into A. oryzae NF1 and thetransformants were grown on MM. Among 95 tested on MMWGH, eighteentransformants did not exhibit DPP IV activity. Six DPP IV negativetransformants were selected and numbered from 8 to 13, and fourtransformants which still exhibited DPP IV activity were numbered from14 to 17. A Southern blot of NcoI digested genomic DNA from these tentransformants was probed with the dppIV PCR fragment (see example 2). Intransformants which did not exhibit DPP IV activity, the 1.5 kb NcoIfragment is absent, which proves that the wild type gene has beenreplaced by the disruption construct. In transformants which retain DPPIV activity, the 1.5 kb fragment is still present, and hybridisingfragments with other molecular weights show that the disruptionconstruct has integrated at another site in the genome.

To quantify DPP IV activity, transformants 10, 11 and 15 as well as A.oryzae NF1 pyrG⁺ transformant were grown for 7 days at 30° C. on liquidMMWG. Enzymatic analyses of the supernatant (table 2) showed thattransformants 10 and 11 had residual proline dipeptidyl-peptidaseactivity, probably due to some non specific enzymes. By contrast,transformant 15 had a higher DPP IV activity (at least 4 times more)compared to the wild type. Inspection of the original screen for DPP IVdisruption mutant revealed additional clones with higher activitycompared to the wild type. Since the disruption construct did notcontain a functional gene, the increase of the activity might have beendue to titration of a repressor.

TABLE 2 DPP IV [mU/ml] LAP [mU/ml] ENDO [mU/ml] NF1 pyrG⁺ 8.7 1.6 2.9Transformant 10 0.4 5.8 3.1 Transformant 11 0.1 6.5 4.5 Transformant 1539.6 5.7 2.7

Example 3

Expression of A. oryzae DPP IV in P. pastoris

Transformation of P. pastoris: plasmid pNFF125 was used as template formultiplying the dppIV gene by PCR. To do so, 200 ng of pNFF125 DNA, 164pmol of oligonucleotides, 120 mM dNTP's were dissolved in 50 ml PCRbuffer (20 mM Tris-HCl pH 8.8, 2 mM MgSO₄, 10 mM (NH₄)₂SO₄, 0.1% TritonX-100, 100 mg/ml nuclease free BSA). A drop of dynawax (Dynazyme) wasadded. To each reaction 2.5 unit of cloned Pfu DNA polymerase(Stratagene) was added in 50 ml of 1×PCR buffer. The A. oryzae dppIVgene was amplified with the primer pair SEQ ID NO:8 and SEQ ID NO:9(these primers covered N- and C- terminal mature protein coding region).The reaction mixtures were subjected to thirty cycles of 1 min 95° C., 1min 44° C. and 3 min 72° C. using Perkin Elmer DNA Thermal Cycler.

The PCR product was digested by EcoRV and NotI and cloned into theSnaBI, NotI digested pKJ115, generating the plasmid pNFF134. P. pastorissphaeroplasts were transformed with 10 μg of pNFF134 linearised by EcoRIas described in the Manual Version 2.0. of the Pichia Expression Kit(Invitrogen).

The P. pastoris expression cassette pKJ115 can insert into the P.pastoris genome via homologous recombination at the alcohol oxidase(AOX1) site and carry, in addition to the cloned coding sequence ofinterest, the his4 gene for selection. Transformants were first selectedon histidine-deficient media (RDB) and then screened for insertion ofthe construct at the aox1 site on minimal methanol plates (MMM).Transformants that were unable to grow on media containing only methanolas a carbon source (BMMY) were assumed to contain the construct in thecorrect yeast genomic location by integration events at the aox1 locusdisplacing of the aox1 coding region. The selected transformants weregrown to near saturation (OD 20 at 600 nm) at 30° C. in 10 ml ofglycerol-based yeast media (BMGY). Cells were harvested and resuspendedin 2 ml BMMY and incubated for 2 days. After two days of incubation, thesupernatant was harvested and 10 ml was analysed by SDS-PAGE accordingto the method of Laemmli (1970) with a separation gel of 7.5% (w/v)polyacrylamide to identify successfully expressing clones. In parallel,the supernatant was checked for activity.

Results show that the obtained concentration of DPP IV was 100 μg/ml.The activity measured in the supernatant was of about 1385 mU/ml. Amongall the transformants, one was deposited under the Budapest Treaty atthe Collection Nationale de Cultures de Microorganismes (CNCM), 25 ruedu Docteur Roux, 75724 Paris, France, on June, where it receives thedeposit number CNCM I-3.

Peptide profiling by size exclusion chromatography (SEC): the efficiencyof DPP IV towards peptides in WG hydrolysates was tested. Enzymes in thesupernatant of dppIV disruptant 11 thus were heat inactivated at 95° C.for 10 min. 140 mU of purified DPP IV produced by P. pastoris CNCM I-3were added to 500 μl of supernatant and incubated at 45° C. up to 24 h.A control experiment without DPP IV addition was performed in parallel.Aliquots were taken at 2 h intervals, acidified with 10% TFA,centrifuged and analysed by SEC on a Superdex Peptide HR 10/30 column(Pharmacia Biotech, Sweden). Separation is based on molecule size ofamino acids and peptides (range: 100-7′000 Da). Chromatography wasperformed under isocratic conditions with 0.1% TFA, 20% acetonitrile inwater at a flow rate of 0.5 ml/min. Detection of amino acid and peptidepeaks was at 215 nm. Peptide and amino acid standards were used tocalibrate the chromatographic system (data not shown).

Results show that an initial increase of small peptides (200-500 Da) canbe detected already after 2 h incubation. Extended incubation (up to 24h) releases more dipeptides. No changes are detected in the controlsample at 2 h and 24 h incubation time. Therefore, it is clear that DPPIV activity liberates dipeptides from wheat gluten hydrolysatesconfirming the efficiency of this enzyme in peptide degradation.

Example 4

Transformation with the Native Promoter of dppIV

The plasmid pNFF126 containing the fragment of 2094 bp ApaI-BamHIencompassing the promotor region and the start of the DPP IV gene (seeSEQ ID NO:1) was introduced into A. oryzae NF1, using pyrG gene asselection marker. The A. oryzae NF1 pyrG⁺ transformants were screened bystaining for their prolyl-dipeptidyl-peptidase activity. Twotransformants (B2, C7) showed a more intensive stain than the otherones. They were therefore cultured onto liquid MMWG for 7 days, 30 ° C.,without shaking, in parallel with three other randomly pickedtransformants and the control A. oryzae NF1 transformed with only pyrG.

The prolyl-dipeptidyl-peptidase activity was analysed from the culturebroths. Results show that transformants B2 and C7 respectively showed afourfold and twofold increase of the prolyl dipeptidyl peptidaseactivity compared to the control, whereas all the other ones do notexhibit any increase of this activity. In the disruption experiment (seeexample 2), also a maximum of fourfold increase of theprolyl-dipeptidyl-peptidase activity was noticed (transformant 15). Thisincrease can be due to a repressor titrated by the multicopies of thepromotor region integrated heterologously in the genome of A. oryzae NF1or by a positive acting factor encoded by the 2094 bp ApaI-BamHIfragment.

Example 5

Functional Derivatives of the DPP IV

Functional derivatives of the DPP IV (SEQ ID NO:2) are preparedaccording to a method adapted from the method described by Adams et al.(EP402450; Genencor). Briefly, the expression cassette pKJ115 containingthe DPP IV was subjected to an in-vitro chemical mutagenesis byhydroxylamine. According to example 3, the mutagenised DNA was then usedto transform P. pastoris. Functional derivatives of the DPP IV,presenting a deletion, addition and/or a substitution of some aminoacids, were finally detected according to their peptide profile obtainedby hydrolysing wheat gluten with purified DPP IV derivatives (seeexample 3).

Examples 6

For preparing a fermented soya sauce, a koji is prepared by mixing anAspergillus oryzae CNCM I-1 koji culture with a mixture of cooked soyaand roasted wheat, the koji is then hydrolysed in aqueous suspension for3 to 8 hours at 45° C. to 60° C. with the enzymes produced duringfermentation of the Aspergillus oryzae CNCM I-1 culture, a moromi isfurther prepared by adding suitable amount of sodium chloride to thehydrolysed koji suspension, the moromi is left to ferment and is thenpressed and the liquor obtained is pasteurized and clarified.

Examples 7

For producing a flavouring agent, a aqueous suspension of a mixture ofcooked soya and roasted wheat is prepared, the proteins are solubilizedby hydrolysis of the suspension with a protease at pH 6.0 to 11.0, thesuspension is heat-treated at pH 4.6 to 6.5, and the suspension isripened with enzymes of a koji culture fermented by Aspergillus oryzaeCNCM I-2.

9 1 5496 DNA Fungus 1 gggccctgag tttaacggtg ctgggtgtgt tattacgcatcatactcttc acccgccttg 60 cagtagttcg gttctattgt caatagctgc tgtcgcaatattctgtcttt tgccaataag 120 gtgaccagga ggggtctttc caggatagat agatggcgacatttatctcg tcgcggcggt 180 gattgtctgt ttgattgatg atgatctctg aaacatgttgaatctggggt acgtaacttg 240 gggtgatcaa ttgacatcca cttagatatg gtacagcaaagtatacctcc tggattctgt 300 gaacaagaat ataaaataag cctcgcgacc gggagtcttgtccctcaaat catcacaatc 360 ccatcgaaca tccgcatcta atttcctcac tcatccttctatccaccgcc aaaatgaagg 420 ccgctaccct cctctctctt ctgagcgtta ccggactcgtcgccgctgct ccagctggca 480 acggtacgta tcctgaacga caatgtaaga cgcttgactgatgattagta ggcccagctg 540 gtggaatcat cgaccgcgat cttcccgtcc ctgtccctggactccctacc aagggtctcc 600 ctattgttga cggattgact ggcggcaata agggtggcgagaagcctgga agcaaggtta 660 ctcctcgtga agaccctacc ggcagcgccc ctgatggcaagggcaatgat ggccccgacg 720 gtgatcttac cggacgtccc ggtcaagggg gtcttgacaaccctttcgat ctccctactc 780 cagagcttcc tcccgtcaag cttcctggcg gacttgacggtggcaagggc ggtctcggcc 840 ttcgtcgtcg tggcagccca gtagacggtc tccctgtcgttgggcctgtt gttggtggtg 900 ttctaggtgg cggtggtgct ggcagtggtg ctggtgccaagggtggtgct ggtagtggta 960 ccgttgggcg tcgtggcagc ccagtagacg gtctccctgttgttgggcct gttgttggtg 1020 gtgtcctagg tggcggtggt gctggcagtg gtgctggtgccaagggtggt gctggtagtg 1080 gtacccctaa gcgccgtgac ggtccagtgg acggtgttcctgtcgttgga gagcttgctg 1140 aaggtgctac tggaggtctt ctaggtggtg atgctggttctgctgatgct gctggtgctg 1200 atgctggtgc tgatgctggt gctggtgctg gtgggcaatagtctaacaag ggctttacgg 1260 catcaatgtg aggttatcca acatccatcc ttggtggccattcgtaaata gcaacaaaga 1320 ggggtggtac ttggtcgcga tgtcattgct cctgcgattgaagctagcga ttcctgtatg 1380 tacaataatt ttaagcacgc ttggttccat actgtttcttcactggtttt tggatatttt 1440 ttcacttatt gaatcttgta gtagtccagc ttctcatggttagacacggg ataacccccc 1500 aatagcatca tctgcaggtt tgatgttgca atggtcaagttttgtcttaa attatgtacg 1560 agtcttgggt taccccgcta gaagctttgc caccaatgaagctgttgctt gtccaacggc 1620 tatcagcggt tttttttatg agaatcttgg caggataggaaaagttggtg gtggtgaagg 1680 agctaatgca ggaggtggag tgactgataa gacgcgatttctgcggggaa aaagaaaaag 1740 gaccaattta tgggactatt tatttaaacg ggaagtcttcaattccgttc gccagccatc 1800 ccttgattcg agctgaactc ggggtttttt ccaccatgaaggtacgtcaa ttccactgat 1860 taaacattat ttgttacata cactccatca ttgagtcaattataattaac acctcataat 1920 tcagtactcc aagcttctgc tgctcctggt cagtgtggtccaggccctgg atgtgcctcg 1980 gaaaccacac gcgcccaccg gagaaggcag taagcgtctcaccttcaatg agaccgtagt 2040 caagcaagca attacgccga cctctcgctc ggtgcaatggctctcgggcg cagaggatgg 2100 atccctacgt gtacgcggcg gaagacggca gtctcaccatcgagaacatc gtcaccaacg 2160 agtcacgcac gctcatcctg cggacaagat tccgacagggaaggaagcgt tcaattactg 2220 gatccatccc gacttgtcgt cggtgctgtg ggcgtccaaccacaccaagc agtatcggca 2280 ttcgttcttt gccgattatt acgtccagga tgtggagtcactcaagtccg tgcccctgat 2340 gcccgatcag gaaggtgata ttcaatatgc ccaatggagccccgtgggca ataccatcgc 2400 ttttgttcgc gagaatgacc tttatgtctg ggataatggtaccgttactc gcattactga 2460 tgatggtggc cccgacatgt tccacggcgt gccggactggatctatgaag aggagatcct 2520 cggcgatcgc tacgcgttgt ggttctcgcc agatggtgaatatctggctt acttgagctt 2580 caatgagact ggggttccga cctacaccgt tcagtattatatggataacc aagagatcgc 2640 tccggcgtat ccatgggagc tgaagataag gtatcccaaggtgtcgcaga cgaatccgac 2700 cgtgacgttg agtctgctta acatcgctag caaggaggtgaagcaggcgc cgatcgacgc 2760 gttcgagtca actgacttga tcattggcga ggttgcttggctcactgata ctcacaccac 2820 cgttgctgct aaggcgttca accgtgtcca ggaccagcaaaaggtcgtcg cggtcgatac 2880 tgcctcgaac aaggctactg tcatcagcga ccgagatgggaccgatggat ggctcgataa 2940 ccttctttca atgaagtata ttggccctat caagccgtccgacaaggatg cctactacat 3000 cgacatctct gaccattcgg gatgggcgca tctgtatctcttccccgttt cgggcggcga 3060 acctatccca ctaaccaaag gcgactggga ggtcacgtctattctgagta ttgatcagga 3120 acgccagttg gtgtactacc tgtcgactca acaccacagcaccgagcgcc atctctactc 3180 cgtctcctat tccacgtttg cggtcacccc gctcgtcgacgacaccgttg ccgcgtactg 3240 gtctgcttcc ttctccgcga actcgggcta ctacatcctcacatacggag gcccagacgt 3300 accctaccag gaactctaca cgaccaacag taccaaaccactccgcacaa tcaccgacaa 3360 cgccaaagta ctcgagcaaa tcaaggacta tgcattgcccaacatcacct acttcgagct 3420 tcccctcccc tccggagaaa ccctcaatgt gatgcagcgcttaccccccg ggttctcccc 3480 ggataagaag taccccatac ttttcacccc atacggcggcccaggcgccc aagaagtgac 3540 caagagatgg caagccctga atttcaaggc ctatgtcgcctccgacagcg aactcgagta 3600 cgtaacctgg actgtcgaca accgcggcac aggtttcaaaggacgcaagt tccgctccgc 3660 cgtcacgcgc caactcggcc tcctcgaagc agaagaccagatctacgccg cgcaacaggc 3720 ggccaacatc ccctggatcg atgcagacca catcggcatctggggctgga gtttcggagg 3780 ctacttgacc agcaaggtcc tggagaagga cagcggtgctttcacattag gagtcatcac 3840 cgcccctgtt tctgactggc gtttctacga ctcaatgtacacggagcgct acatgaagac 3900 cctctcgacc aatgaggagg gctacgagac cagcgccgtccgcaagactg acgggttcaa 3960 gaacgtcgag ggcggattct tgatccagca cggaacgggcgacgataacg tccatttcca 4020 gaactcggct gcgctggtgg atctcctgat gggcgatggcgtctctcctg agaagctcca 4080 ttcgcaatgg ttcacagact cagaccacgg aatcagctaccatggtggcg gcgtgttcct 4140 gtacaagcaa ctggcccgga agctctacca ggagaagaaccgacagacgc aggtgctgat 4200 gcaccagtgg actaagaagg acttggagga gtagaagcggcacatcattc attcatttta 4260 aagcgactgg ctacacatag catacatagc aattgatacttcgtatttta ccctccccac 4320 agccacgacc atcacccatt ggcgcaaaat tctccccgcaccataaacta gcgcgacgag 4380 gctgaaaatc tgccagaaat ctacttaaag ctcgtgttggcccagtccct cacaacccaa 4440 accatcccaa gtaaacaaaa ccaaaaaaaa atcccatagaaaatggccga catccccacc 4500 tcaacagtcc aaatcacaac cctccccacc aaatccgtaacaatcacccc gcaacgagcg 4560 accatcgttc gcgagataca cacctccatc caggtatgcacataccacct cacctgacca 4620 tccaacccta cttacagtca acgtaaacta acaaaattaaaaaaataaaa agacaggcca 4680 acacgaacta ataatcaccg gcctcgaccc aagagtagacaccgactcca ttttactcga 4740 aggaacagga acggccacaa taaccgatat ccaaacctcgatagtccccc gacaggaaaa 4800 attcgaggat atctatcccg ccgaatcaga ctccgacgactccccagagc ccgattccga 4860 ctccgacctt gaccacgatg accccgagtt acaagctatctccgcatcca tagccgaagt 4920 cgaagcgcga cttgcgcgag cggaaaatga acagacgatggcggtttcca tccgggagtt 4980 tctggatggg tatgccaaga agatggatcc ggagcatgtggacgcggaga tgctagatgg 5040 gttcttgggg ctttataccc ggcagcgggt ggaggggtttcagcggcatc atcaggctgg 5100 ggtggagtat gggaaggggg agagggagct tgcgcggttggtgaagagga acgcgggaag 5160 attgagggtc ggttgaagag ggctagggag gtggtgaagaagaaggagcg gagggagagg 5220 gagaagagag ccaccgagcg tgcgaggaag actgaacagcggaagatgaa gagggaggag 5280 agactcaagt tctggacgac gcgggttggg caggtggttgtgtcatctgg atagtcaggc 5340 cgggactwgc cggcgcagtt ccatcgttga atcgggttgaacggttktct ggttgtgtgt 5400 agtatttcat gcggagcctg tgtggatgtc gacgtgtgcgtgctgagact atgttgtgta 5460 cgwmtataga tttaattaag gatcckgcgt gccgcc 54962 771 PRT Fungus 2 Met Lys Tyr Ser Lys Leu Leu Leu Leu Leu Val Ser ValVal Gln Ala 1 5 10 15 Leu Asp Val Pro Arg Lys Pro His Ala Pro Thr GlyGlu Gly Ser Lys 20 25 30 Arg Leu Thr Phe Asn Glu Thr Val Val Lys Gln AlaIle Thr Pro Thr 35 40 45 Ser Arg Ser Val Gln Trp Leu Ser Gly Ala Glu AspGly Ser Leu Arg 50 55 60 Val Arg Gly Gly Arg Arg Gln Ser His His Arg GluHis Arg His Gln 65 70 75 80 Arg Val Thr His Ala His Pro Ala Asp Lys IlePro Thr Gly Lys Glu 85 90 95 Ala Phe Asn Tyr Trp Ile His Pro Asp Leu SerSer Val Leu Trp Ala 100 105 110 Ser Asn His Thr Lys Gln Tyr Arg His SerPhe Phe Ala Asp Tyr Tyr 115 120 125 Val Gln Asp Val Glu Ser Leu Lys SerVal Pro Leu Met Pro Asp Gln 130 135 140 Glu Gly Asp Ile Gln Tyr Ala GlnTrp Ser Pro Val Gly Asn Thr Ile 145 150 155 160 Ala Phe Val Arg Glu AsnAsp Leu Tyr Val Trp Asp Asn Gly Thr Val 165 170 175 Thr Arg Ile Thr AspAsp Gly Gly Pro Asp Met Phe His Gly Val Pro 180 185 190 Asp Trp Ile TyrGlu Glu Glu Ile Leu Gly Asp Arg Tyr Ala Leu Trp 195 200 205 Phe Ser ProAsp Gly Glu Tyr Leu Ala Tyr Leu Ser Phe Asn Glu Thr 210 215 220 Gly ValPro Thr Tyr Thr Val Gln Tyr Tyr Met Asp Asn Gln Glu Ile 225 230 235 240Ala Pro Ala Tyr Pro Trp Glu Leu Lys Ile Arg Tyr Pro Lys Val Ser 245 250255 Gln Thr Asn Pro Thr Val Thr Leu Ser Leu Leu Asn Ile Ala Ser Lys 260265 270 Glu Val Lys Gln Ala Pro Ile Asp Ala Phe Glu Ser Thr Asp Leu Ile275 280 285 Ile Gly Glu Val Ala Trp Leu Thr Asp Thr His Thr Thr Val AlaAla 290 295 300 Lys Ala Phe Asn Arg Val Gln Asp Gln Gln Lys Val Val AlaVal Asp 305 310 315 320 Thr Ala Ser Asn Lys Ala Thr Val Ile Ser Asp ArgAsp Gly Thr Asp 325 330 335 Gly Trp Leu Asp Asn Leu Leu Ser Met Lys TyrIle Gly Pro Ile Lys 340 345 350 Pro Ser Asp Lys Asp Ala Tyr Tyr Ile AspIle Ser Asp His Ser Gly 355 360 365 Trp Ala His Leu Tyr Leu Phe Pro ValSer Gly Gly Glu Pro Ile Pro 370 375 380 Leu Thr Lys Gly Asp Trp Glu ValThr Ser Ile Leu Ser Ile Asp Gln 385 390 395 400 Glu Arg Gln Leu Val TyrTyr Leu Ser Thr Gln His His Ser Thr Glu 405 410 415 Arg His Leu Tyr SerVal Ser Tyr Ser Thr Phe Ala Val Thr Pro Leu 420 425 430 Val Asp Asp ThrVal Ala Ala Tyr Trp Ser Ala Ser Phe Ser Ala Asn 435 440 445 Ser Gly TyrTyr Ile Leu Thr Tyr Gly Gly Pro Asp Val Pro Tyr Gln 450 455 460 Glu LeuTyr Thr Thr Asn Ser Thr Lys Pro Leu Arg Thr Ile Thr Asp 465 470 475 480Asn Ala Lys Val Leu Glu Gln Ile Lys Asp Tyr Ala Leu Pro Asn Ile 485 490495 Thr Tyr Phe Glu Leu Pro Leu Pro Ser Gly Glu Thr Leu Asn Val Met 500505 510 Gln Arg Leu Pro Pro Gly Phe Ser Pro Asp Lys Lys Tyr Pro Ile Leu515 520 525 Phe Thr Pro Tyr Gly Gly Pro Gly Ala Gln Glu Val Thr Lys ArgTrp 530 535 540 Gln Ala Leu Asn Phe Lys Ala Tyr Val Ala Ser Asp Ser GluLeu Glu 545 550 555 560 Tyr Val Thr Trp Thr Val Asp Asn Arg Gly Thr GlyPhe Lys Gly Arg 565 570 575 Lys Phe Arg Ser Ala Val Thr Arg Gln Leu GlyLeu Leu Glu Ala Glu 580 585 590 Asp Gln Ile Tyr Ala Ala Gln Gln Ala AlaAsn Ile Pro Trp Ile Asp 595 600 605 Ala Asp His Ile Gly Ile Trp Gly TrpSer Phe Gly Gly Tyr Leu Thr 610 615 620 Ser Lys Val Leu Glu Lys Asp SerGly Ala Phe Thr Leu Gly Val Ile 625 630 635 640 Thr Ala Pro Val Ser AspTrp Arg Phe Tyr Asp Ser Met Tyr Thr Glu 645 650 655 Arg Tyr Met Lys ThrLeu Ser Thr Asn Glu Glu Gly Tyr Glu Thr Ser 660 665 670 Ala Val Arg LysThr Asp Gly Phe Lys Asn Val Glu Gly Gly Phe Leu 675 680 685 Ile Gln HisGly Thr Gly Asp Asp Asn Val His Phe Gln Asn Ser Ala 690 695 700 Ala LeuVal Asp Leu Leu Met Gly Asp Gly Val Ser Pro Glu Lys Leu 705 710 715 720His Ser Gln Trp Phe Thr Asp Ser Asp His Gly Ile Ser Tyr His Gly 725 730735 Gly Gly Val Phe Leu Tyr Lys Gln Leu Ala Arg Lys Leu Tyr Gln Glu 740745 750 Lys Asn Arg Gln Thr Gln Val Leu Met His Gln Trp Thr Lys Lys Asp755 760 765 Leu Glu Glu 770 3 18 DNA Fungus 3 gcctggacca cactgacc 18 418 DNA Fungus 4 tccaccatga agtactcc 18 5 18 DNA Fungus 5 atcgccgaggatctcctc 18 6 23 DNA Fungus 6 gaattccatg gtgtcctcgt cgg 23 7 24 DNAFungus 7 gaattcgagc cgtcagtgag gctc 24 8 32 DNA Fungus 8 tggtcgatatcctggatgtg cctcggaaac ca 32 9 30 DNA Fungus 9 ttgcggccgc tactcctccaagtccttctt 30

What is claimed is:
 1. A recombinant prolyl-dipeptidyl-peptidase (DPPIV) from Aspergillus oryzae comprising the amino-acid sequence fromamino acid 1 to amino acid 755 of SEQ ID NO:2 or polypeptides having atleast 80% identity to amino acids 1-755 of SEQ ID NO:2 havingprolyl-dipeptidyl peptidase activity.
 2. A recombinantprolyl-dipeptidyl-peptidase according to claim 1 which is fused to aleader peptide.
 3. A recombinant prolyl-dipeptidyl-peptidase accordingto claim 2 which is fused to the leader peptide of Aspergillus oryzaeDDP IV having the amino-acid sequence from amino acid-16 to amino acid-1of SEQ ID NO:2 or variants thereof having at least 80% identity to aminoacids −16 to −1 of SEQ ID NO:2, wherein said variants are able tointegrate the DPP IV into the cell wall or the cell membrane or secretethe enzyme into the periplasmic space or into the culture medium.
 4. Aleader peptide of Aspergillus oryzae DPP IV having the amino-acidsequence from amino acid-16 to amino acid−1 of SEQ ID NO:2 or variantsthereof having at least 80% identity to amino acids −16 to −1 of SEQ IDNO:2, wherein said variants are able to integrate the DPP IV into thecell wall or the cell membrane or secrete the enzyme into theperiplasmic space or into the culture medium.
 5. An isolated DNAmolecule which comprises a dppIV gene encoding DPP IV according to claim1.
 6. An isolated DNA molecule according to claim 5, which is a vectorcomprising the dppIV gene.
 7. An isolated DNA molecule according toclaim 5, wherein the dppIV gene is operably linked to at least oneregulatory sequence able to direct the expression of the gene.
 8. A DNAmolecule according to claim 7, wherein the regulatory sequence isderived from another organism than the one from which the dppIV gene isderived.
 9. An isolated DNA molecule according to claim 5, wherein dppIVgene comprises the coding parts of the nucleotide sequence SEQ ID NO:1or variants thereof encoding polypeptides having at least 80% identifyto amino acids 1-755 of SEQ ID NO:2 having prolyl-dipeptidyl peptidaseactivity.
 10. A cell which expresses DPP IV according to claim 1 byrecombinant technology.
 11. A cell according to claim 10, which isPichia pastoris CNCM I-1886.
 12. A cell according to claim 10 which isable to over-express DPP IV.
 13. A cell according to claim 12, which isAspergillus oryzae capable of providing at least 50 mU ofprolyl-dipeptidyl-peptidase activity per ml of supernatant when grown ina minimal medium containing 1% (w/v) of wheat gluten.
 14. An Aspergillusoryzae which is able to over-express the enzyme according to claim 1 byrecombinant technology wherein multiple recombinant functional dppIVgenes have been integrated.
 15. An Aspergillus oryzae according to claim14 which is the Aspergillus oryzae CNCM I-1888.
 16. An Aspergillus whichhas integrated multiple copies of the promoter having the codingnucleotide sequence from nucleotide 1836 to nucleotide 1966 of SEQ IDNO:1.
 17. An Aspergillus oryzae according to claim 16, which is theAspergillus oryzae CNCM I-1887.
 18. An Aspergillus naturally providing aprolyl-dipeptidyl-peptidase activity which is manipulated genetically sothat dppIV gene is inactivated.
 19. A method for producing a recombinantprolyl-dipeptidyl-peptidase (DPP IV) from Aspergillus oryzae comprisingthe amino-acid sequence from amino acid 1 to amino acid 755 of SEQ IDNO:2 or polypeptides having at least 80% identity to amino acids 1-755of SEQ ID NO:2 having prolyl-dipeptidyl peptidase activity comprisingcultivating recombinant cells according to claim 10 in a suitable growthmedium under conditions that the cells express the enzyme, andoptionally isolating the enzyme in the form of a concentrate.